This project studies the regulation of expression of genes encoding lens fiber membrane channel proteins, which are essential for maintaining the transparency and correct refractive index of the lens. We are presently focusing on the regulation of expression of the gene encoding MIP/AQP0, the major intrinsic protein of the lens fiber membrane that is specifically expressed in the ocular lens. We study the MIP gene regulatory elements and the signaling pathways responsible for the activation of the MIP promoter in FGF2-induced differentiation of explanted lens epithelia into fibers. Our results indicate that the MIP gene 5'-flanking sequence contains regulatory elements required for MIP gene expression in differentiating lens cells that are responsible to FGF2. We are currently mapping the FGF2 signaling pathways involved in the activation of the MIP promoter in the lens fibers. Lens Major Intrinsic Protein (MIP)/aquaporin 0, the major intrinsic protein of the lens fiber membrane, belongs to an ancient family of transmembrane channel proteins and plays an important role in lens transparency. MIP/Aquaporin 0 functions as a water chanel. However, it may have additional functions in the lens to maintain lens transparency. Our goal is to identify and characterize proteins that interact with MIP using the yeast two-hybrid system, to elucidate the role of these interactions in MIP functions. We have identified lens cDNA clones encoding proteins that interact with MIP in yeast cells: gamma E-crystallin and ribosomal protein L4. We are further characterizing the interaction of MIP with gamma E-crystallin in mammalian cells and mapping the amino acid domains of MIP and gamma E-crystallin involved in this interaction. Further experiments will allow us to confirm whether these protein-protein interactions have any physiological relevance to MIP functions.